Quality Control of 11 Cannabinoids by Ultraperformance Liquid Chromatography Coupled with Mass Spectrometry (UPLC-MS/MS)

Objective Cannabinoid extraction from Cannabis sativa L. (hemp) for nonmedical purposes has become popular in the United States. Concerns, however, have been raised regarding the accuracy of the labels for cannabinoid levels in the commercial products. Methods In this study, we developed rapid, sensitive, selective, accurate, and validated liquid chromatography-tandem mass spectrometry for the quantification of cannabinoids. The methods are for determining 11 cannabinoids in cannabis (hemp) extracted in oil form, and we investigated the accuracy of the labeling and thermal stability regarding the cannabinoids on 17 oil cannabis samples. Results In the UPLC chromatogram, we see a good resolution and there is no matrix effect and the accuracy were 98.2% to 102.6%, and the precision was 0.52%–8.18%. The linearity of the calibration curves in methanol was with a regression r2 ≥ 0.99. The lowest of detection (LOD) was 5–25 ng/mL, and the limit of quantification (LOQ) was 10–50 ng/mL. The study showed that only 30% of the commercial samples were within the acceptable range of +/−10% compared to the labeled ingredient concentrations. The thermal stability test profile showed a change in the concentration of cannabinoids in each sample at 37°C for one week, with an average loss of cannabinoids up to 15%. Conclusion The validated method proved to be selective, accurate, and precise, with acceptable linearity within the calibration range with no matrix effect. The stability profile data indicated that high temperatures could change the quality of commercial samples.


Introduction
Cannabis sativa (marijuana) products are widely consumed products for recreational purposes nationwide, and using as medicinal forms is currently under scrutiny [1][2][3]. Delta-9tetrahydrocannabinol (Δ 9 -THC) is the primary psychoactive compound in cannabis preparations and is converted to other analogs by several enzymes in the liver and gut microbiota [4,5]. Cannabinoids are a class of chemical compounds synthesized in plants by a very complex enzymatic system that converts one analog to another. For example, cannabigerolic acid (CBGA) is converted to cannabigerol (CBG) through decarboxylation ( Figure 1) [6,7]. Despite research breakthroughs over the last three decades, cannabis plants remain classifed by the Food and Drug Administration (FDA) as a Schedule I Controlled Substance under the Controlled Substances Act of 1970. In states that have not passed medicinal marijuana or recreational marijuana legislation but do allow hemp products for commercial sale, Δ 9 -THC must be at or below the concentration of 0.3% [8][9][10]. Cannabidiol (CBD) and Δ 9 -THC are isolatable phytocannabinoid molecules used to treat cancer [11,12].
Cannabis sativa L (cannabis, hemp, or marijuana) is subspecies hybrid, and the extracts are generally classifed into two main categories: full-and broad-spectrum products. Broad-spectrum products contain primary cannabinoids with various concentrations extracted from cannabis and have bioactivity or therapeutic efects. Te full-spectrum extract contains primary cannabinoids such as Δ 9 -THC at the level below the detection level for most laboratory testing purposes. Te main active ingredient in these products is CBD. CBD is white to slightly of-white in color, crystalline, and has a mild (almost unnoticeable) smell/odor. CBD can be obtained by extraction and distillation from plants such as Cannabis sativa via inforescence or synthesized by stereoselective laboratory techniques. CBD-containing products have emerged in high demand in many states since they are marketed in herbal, cosmetic, and pseudopharmaceutical forms. Tese products use current good manufacturing practices (GMPs/CGMPs) and have been considered to validate the concentrations of CBD and Δ 9 -tetrahydrocannabinol (Δ 9 -THC) in diferent products ranging among topical oils, tinctures, gummies, soft-gel lozenges, and smokables [13][14][15].
Tis study aims to develop a robust ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) quantitative laboratory analysis of cannabinoids. Such analysis is paramount for quality control processes that can be used to quantify and verify cannabinoid levels in commercially available products. Moreover, the efects of temperature were investigated to quantify the changes in cannabinoid levels.

Instrumentation and Data
Processing. High-performance liquid chromatography-tandem mass spectrometry was used. Te Acquity Classic UPLC ® system consisted of a Waters sample manager, binary solvent manager, cooled sample trays, integrated column heater, and degasser. Te UPLC system was equipped with binary system pumps, an autosampler, a built-in degasser, and a column heater coupled with a Xevo TQ MS detector. A sample loop in the injection mode was used to inject 10 µL samples. MassLynx software (version 4.2) was used to collect the data processed using TargetLynx (version 4.2). Cannabinoids in eluted samples were quantifed by using a UPLC-MS/MS system consisting of a quadrupole time-of-fight mass spectrometer system (Q-Tof-MS/MS) (Waters Xevo TQ-XS with Z-spray ionization and step wave source optimization). Te equipment calibration and detector validation processes were performed daily using octreotide and standard solutions with the integrated Intelli Start procedure of the MassLynx V4.2 system software. Te resulting mass spectrometric parameters were determined using argon collision gas for collision-induced dissociation (CID), coupled with the exact mass measurement with time-offight (ToF) with tandem mass spectrometry (MS/MS) transitions where the analytes and standards were monitored in the positive or negative ion modes. Te exact mass was used to determine the elemental composition of the target molecules.

Liquid Chromatographic (LC) Conditions.
Analytes were separated on an Aquity UPLC BEH C18 analytical column (2.1 × 100 mm, 1.7 µm particle size, and 130 Ǻ pore size) preceded by an Acquity UPLC BEH C18 VanGuard precolumn (2.1 × 5 mm, 130 Ǻ). Te fow rate was kept at 0.5 mL/min, and 5 µL of the sample was injected into the column. Te autosampler was maintained at 10°C throughout the analysis, and the analytical column was maintained at 45°C. Te mobile phases consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). A linear gradient was used to separate the analytes over a run time of 13 min. Te gradient conditions were as follows: 50% A for 1 min; 100% B for 8 min; 50% B for 3 min; and equilibration of the column for 1 min.

Mass Spectrometry Conditions.
Quadrupole time-offight tandem mass spectrometer system (Waters SYNAPT G2-Si Q-ToF) parameters were optimized using tandem MS (MS/MS) ions for each standard solution of cannabinoids in the positive and negative modes. Te most common cannabinoids have similar precursor (parent) ions but diferent products (daughters). Te method was validated and followed the FDA guidelines. Electrospray ionization (ESI) in the positive and negative modes was used to quantify the analytes' tandem MS/MS transitions. Major analyte-specifc mass spectrometer settings used during the analysis in the positive mode for protonated precursors (M + H) + were selected for CBD, CBG, CBDV, THCV, CBN, Δ 8 -THC, Δ 9 -THC, and CBC. Te deprotonated precursors (M-H) − were chosen for CBDA, Δ 9 -THCA, and CBGA. A total ion chromatogram (TIC) was used to quantify the analytes (Table 1). Mass spectrometry parameters included capillary voltage of 1.50 kV, collision gas fow of 0.15 mL·min −1 , extractor voltage of 3 V, desolation temperature of 500°C, source temperature of 150°C, and desolation gas fow of 1000 L/h, and the scan MS was 50-1200 m/z. Te quantifcation was operated in the MSMS mode. For MS E experiments, one acquisition function with diferent collision energy ramps was used for additional MS/MS experiments with electrospray ionization (ESI). Te system was organized with the Analyst 1.6.3 software, and data were collected by MultiQuant 3.0.2 system. Data were processed using TargetLynx software (within MassLynx).

Validation of the Bioanalytical Method.
Tis validation method followed the general guidelines for bioanalytical method development issued by the US FDA [17]. Te limits of detection and quantifcation, linearity, precision, accuracy, recovery, and matrix efect tests were evaluated and validated. Oil of English ivy plant (0% CBD) was used as the matrix to measure the recovery percentages.

Standard and Quality Control Samples.
A standard stock solution of the 11 cannabinoid solutions was prepared in methanol at a 1 mg/ml concentration. Calibration curves and quality control samples were included for each run. Te area under curve (AUC) ratios were recorded and plotted against the concentrations of the standards. Five replicates were used for each of the six stock solutions of each of the 11 cannabinoids with the fnal concentrations of 0, 50, 100, 150, and 200 ng/ml. All spiked samples and stock solutions were stored at −20°C. Te lowest of detection (LOD) was used where the signal-to-noise ratio, S/N, was higher than 3, whereas the limit of quantifcation (LOQ) was established at a signal-to-noise ratio S/N ≥ 10. Te coefcient of variation (CV%) was ≤20%.
Te acceptance criteria for quality control samples (QCs) include the limit of quantifcation (LOQ), the middle of quantifcation or detection (MOQ/MOD), and the highest of quantifcation or detection (HOQ/HOD) at RSD ≤ 15%.

Extraction
Procedure. Samples were extracted from cannabis using the solid-liquid method. Te weights of the cannabis samples, such as fower, crude extract, tincture, or cream on clean and dry paper, were 0.1-0.5 g. Te fower sample was ground into a fne powder using a mortar and pestle. Five milliliters of acetonitrile (LCMS grade) were added to the sample in the centrifuge tube. Gen Power 125 was used to mix the powder with the solvent for 20 min and then the mixture was vortexed for 3 min. Te mixture was sonicated for 15 min and centrifuged at 13000 rpm for 15 min. Te supernatant was then transferred into a separate tube. Te extraction with ACN was repeated 4 times, and all the fractions (20 ml) were mixed. Te extract went through a dilution factor of 100 and was vortexed for 1 min and was then fltered using a 0.45 µm PTFE flter unit. Samples were stored at 4°C for analysis. Before transferring 100 µL of the extract to the LCMS vial, the extract was centrifuged for 5 min.

Matrix Efect, Recovery, Accuracy, and Stability Tests.
For analysing matrix efects, the oil of English ivy plant was used as the matrix with 0% cannabinoids. Te AUCs of 11 standard cannabinoids were spiked and quantifed and compared to the spiked solvent (ACN) at the three quality control concentrations. Te AUC was also used to measure the recovery percentages after extraction. In fve replicates, accuracy was evaluated relative to the calibration curve at three diferent concentrations (LOQ, MOC, and HOQ). Te efect of temperature on the stability of the samples was investigated for all the analytes. After seven consecutive days, the concentration changes were recorded at diferent temperatures (−20, 4, 25, 37°C). Te experiments were replicated (n � 5). Figure 1 shows the schematic presentation of the eleven cannabinoids analyzed in this report, and Table 1 lists their retention times (RTs). Figure 2 shows the chromatograms of water spiked with cannabinoids at a LOQ concentration of 5 ng/ml where the  MS mode of detection was positive (2(a)) or negative (2(b)); m/z(s) were determined by UPLC-MS/MS, and the chemical structure is also shown in Table 1. Te degree of the linearity for the calibration curve was within the acceptable range (r 2 � 0.99) [18] (Table 1).

Precision and Accuracy.
Te precision and accuracy of the method were measured by analyzing data of the LOQ, MOQ/MOD, and the HOQ/HOD of the 11 cannabinoids. Tese were prepared and evaluated using fve replicate points within the calibration curve between 50 and 200 ng/mL. Te correlation coefcient (r 2 ) was determined to be ≥ 0.99 ( Figure S1).

Extraction Recovery and the Matrix Efect.
Te method assessed the extraction recovery and the matrix efect of 11 cannabinoid analytes at LOQ, MOQ/MOD, and HOQ/HOD for all analytes (n = 5). Te extraction recovery ranged from 86.0 to 110.88%. Te matrix efect was detected for the 11 cannabinoids with 3 replicates in the range of 91.98-111.44% (Table S1, Figure 3). Table 2 lists the comparison between the experimental and labeled cannabinoid concentration for 17 commercially available products. Te assay quantifed the cannabinoids in each of these commercial products. Tables 2 and 3  Regarding the calculation of the concentrations, it is important to clarify that the concentrations were determined using the calibration curves generated in the assay. Te calibration curves were created based on the best-ft linear regression method, as shown in Figure S1. To calculate the cannabinoid concentrations in the samples, the software (TargetLynx) integrated within MassLynx was employed. Te software utilizes the calibration curves to determine the concentration of cannabinoids in the samples based on their respective peak areas. Tis method allows for accurate quantifcation of cannabinoids by incorporating the calibration curves developed using the UPLC-MS/MS method. Te label contained the concentration of the major cannabinoids in these products, ranging from 1.3 to 95 mg/mL, indicating a high percentage of error (% diference) ( Table 2) in some cases. Te US Pharmacopeia (USP) guidelines suggest that the experimental results should be within +/−10% of the reported data in the product. Tese products' thermal stability in terms of the level of cannabinoids was assessed as they were stored at diferent temperature conditions as follows: −20, 4, 25, and 37°C for one week (Table 3). Figure 1 shows that the method was very sensitive compared to published reports [19,20]. Table 1 also lists the LOD for the 11 cannabinoids. Te peak intensities and UAC of the analytes were the same whether the acquired chromatogram was obtained in the blank or matrix conditions. Table 2 presents the comparison between the labeled cannabinoid concentrations and the experimental concentrations obtained through quantifcation using the UPLC-MS/MS method for 17 commercially available cannabinoid products. Te labeled concentrations of major cannabinoids in these products were provided by the manufacturer, ranging from 1.3 to 95 mg/mL. Te percent diference (% diference) between the labeled and experimental concentrations is also reported in the table. It is important to note that the US Pharmacopeia (USP) guidelines suggest that experimental results should fall within +/−10% of the reported data on the product labels. Based on this guideline, several products in Table 2 show a high percentage of error (% diference) between the labeled and experimental concentrations. For example, sample 2 (THC) exhibits a −60.0% diference, indicating a lower experimental concentration compared to the labeled value. On the other hand, samples 3, 4, 5, and 13 (CBD and THC) show positive percent diferences, indicating higher experimental concentrations than the labeled values. Regarding sample 10, it is identifed as a noncannabinoid sample in Table 2, which explains why the experimental concentration is reported as 0.0 mg/mL. Te presence of a noncannabinoid sample in the dataset can provide valuable information for assessing the specifcity and accuracy of the quantifcation method, as it should ideally yield a nondetectable result.

Discussion
Te accuracy and precision data were within the acceptance criteria, with a precision of ≤15% and accuracy within ±15%. Te actual accuracy of diferent analytes, shown in Table 4, was between 98.29 and 110.27% of their points for calibrators. Te precision was between 0.52 and 8.18% (Table 4).
Likewise, no matrix efect was detected, and it was within the acceptable range for the 11 cannabinoids. Table 1 indicates that only 30% of the samples were within the acceptable range. Our explanation for samples whose experimental values did not match the labels is human error in their analysis or inadequate storage and/or transportation conditions. Te results (Table 3) showed that at 37°C, CBD and THC concentrations could change by more than 10% on average. Temperature is a signifcant factor that can change the concentrations of some isomers or acid forms of cannabinoids. For example, oxidation and reduction may convert Δ 9 -THCA to CBNA and Δ 9 -THC to Δ 8 -THC, and decarboxylation processes may convert CBGA to CBG, CBDA to CBD, and Δ 9 -THCA to Δ 9 -THC20 (Figure 1).

Conclusion
With the increase in consuming cannabis (hemp) products in the market, we developed a new analytical method to analyze cannabinoid-containing commercial products to determine whether they meet the current regulatory requirements to protect consumers' health. UPLC-MS/MS has become a successful technique for analyzing and measuring cannabinoids with high sensitivity and precision. Te used UPLC-MS/MS in our study was developed and validated by the FDA. Te validation met the acceptance criteria, including sensitivity, speed of analysis within 13 min, accuracy, precision, and recovery. Chromatographic separation and ion extraction by MS are powerful tools with good sensitivity and resolution for quantifying 11 cannabinoids. Te lowest of the quantitation reported was very sensitive to low concentrations of the 11 tested cannabinoids (∼5 ng/ mL). Te labels of seventeen cannabinoid-containing commercial samples were investigated using our validated LC-MS/MS method. Te results showed that only 30% of the samples met the acceptance range. Our temperature-stability tests indicate that 4°C is a good standard temperature to maintain the cannabinoid products under safe conditions. Te temperature in combination with humidity, light, packaging materials, and excipient materials will be the subject of our future investigation on cannabinoid products such as oil, vapor, cream, tincture, and cigarettes to increase the standardized laboratory testing for the quantifcation of all cannabis products.

Data Availability
Te data used to support the fndings of this study are included with the supplementary information fles..

Conflicts of Interest
Te authors declare that they have no conficts of interest.  Journal of Analytical Methods in Chemistry 7 Pharmacy for their valuable insights and contributions during the conceptualization and methodology phases of the study. Figure S1: calibration curves for 11 diferent target cannabinoids. In our study, we established calibration curves for each of the 11 target cannabinoids using a best-ft linear regression approach for quantifcation. Te calibration curves were constructed based on known concentrations of standard solutions of each cannabinoid. Tese calibration curves served as a crucial reference for accurately determining the concentrations of cannabinoids in the samples analyzed. Table S1: matrix efect and recovery efect of cannabinoids in samples. Table S1 presents the matrix efect and recovery efect of various cannabinoids in the samples, with each value representing the mean percentage (n = 5) obtained during the analysis. Te matrix efect indicates the interference of the sample matrix on the analyte's response, while the recovery efect represents the efciency of the extraction method in quantifying the cannabinoids accurately. For each cannabinoid, three levels of concentration were considered: LOQ, limit of quantifcation; MOQ, midpoint of quantifcation; and HOQ, highpoint of quantifcation. (Supplementary Materials)